Project Summary The goal of my research is to delineate how HIV modulates cellular ESCRT proteins to facilitate budding and release. Current FDA-approved anti-retrovirals target early events or virion morphogenesis but not late events. In this application, I propose to delineate a novel regulation of host ESCRT adaptor protein ALIX, by a proinflammatory ?-chemokine, CCL2, which we have shown facilitates efficient virus release. Our preliminary results indicate that CCL2 secreted by clade B HIV-infected cells stimulates HIV-1 replication. This stimulation requires the LYPXnL motif in Gag-p6 and ALIX protein in the cell. CCL2 treatment mobilized ALIX from Actin stress fibers to the cytoplasm, while CCL2 depletion increased ALIX localization to Actin. These findings, for the first time, show that a chemokine induced by HIV is exploited by HIV-1 to promote its own replication by manipulating ESCRTs. Clade C HIV-1 neither induces CCL2 production nor contains a LYPXnL motif due to a conserved LY dipeptide deletion. It is recently reported that, clade C isolates acquire tetrapeptide insertions, PYRE, PYKE or PYQE (collectively termed PYxE insertions) in Gag-p6 replacing the missing LY dipeptide. Our results show that these insertions enhance HIV-1C release efficiency. We propose to investigate the link between LYPXnL motif, ALIX regulation and the mechanism of PYxE-insertion mediated increase in viral fitness through two specific aims. In Specific Aim 1, we propose to delineate the mechanistic basis of CCL2-mediated ALIX regulation. In order to better define the dynamic movement of ALIX between Actin stress fibers and cytoplasmic compartments in response to varying CCL2 levels, we will employ confocal imaging of HeLa cells, macrophages and CD4 T cells that are untreated, CCL2 treated or anti-CCL2 treated. We will determine the time course of ALIX movement upon CCL2 modulation, quantify the ALIX-Actin association and measure the changes in the actin stress fiber formation. Furthermore, in order to understand what triggers the dynamic movement of ALIX in response to CCL2, we will exploit the stark differences in soluble ALIX levels in the presence and absence of anti-CCL2 to identify post-translational modifications on both the soluble and Actin-associated ALIX. In Specific Aim 2, we will address the biological role of PYxE insertions recently reported to be observed in the Gag-p6 of HIV-1C isolates and found to increase virus release efficiency in our laboratory. We will investigate why the PYxE insertions in HIV-1C Gag-p6 improve virus release efficiency and potentially an improved replication fitness. We will first test whether PYxE insertions restore the ability of HIV-1C Gag-p6 to bind ALIX by measuring the Kd of binding. We will construct molecular clones of HIV-1C bearing each of these insertions and examine if the increased virus budding observed is associated with an increased virus fitness. We will also examine the ALIX requirement for budding of such PYxE insertion viruses by silencing Alix expression and finally we will examine whether HIV-1C with the insertions responds to CCL2 by increased virion budding and replication fitness.